My proposal focuses on the unrecognized role of endo-lysosomal transport machinery in regulating synaptic vesicle protein levels. The endosomal adaptor protein AP-3 selectively recognizes certain synaptic vesicle proteins, including metal and neurotransmitter transporters. Decreased levels of these transporters in neuronal AP-3-deficient mice result in epilepsy. However, these transporters increase in synaptic vesicles from ubiquitous AP-3-deficient mice, suggesting that AP-3 isoforms together balance the levels of synaptic vesicle proteins to maintain proper neurotransmission. Specifically, I hypothesize that ubiquitous AP-3 incorporates lysosomal fusion machinery into neuronal AP-3-derived synaptic vesicles, allowing for lysosomal delivery and degradation of synaptic vesicle proteins. This mechanism would prevent the aberrant accumulation of synaptic vesicle proteins associated with neurodegenerative disorders. Therefore, my research proposal addresses the existence and membrane transport dynamics of this novel synaptic/lysosomal vesicle as a mechanism for delivery of synaptic vesicle membrane proteins to lysosomes for degradation. In support of an AP-3-derived synaptic/lysosomal vesicle, the AP-3 microvesicle proteome identified both synaptic vesicle and lysosomal membrane proteins. To further test this hypothesis, the first aim addresses whether AP-3-sorted synaptic vesicle and lysosomal membrane proteins reside in the same vesicles by using biochemical techniques, including subcellular fractionation and vesicular isolation, as well as quantitative deconvolusion microscopy and immuno-gold electron microscopy, in both neuronal cell lines and primary culture neurons. The second aim addresses the contribution of -1) AP-3-dependent vesicle biogenesis, by using AP-3-deficient mouse brains to examine vesicle content and 2) vesicle fusion with lysosomes through the AP-3-sorted fusion machinery, namely VAMP7 and vps33b - to the half-life of synaptic vesicle proteins; I will use siRNA knockdown of the AP-3-sorted lysosomal fusion machinery, VAMP7 and vps33b, in primary neurons and neuronal cell lines and perform pulse chase experiments to determine synaptic vesicle membrane protein half-life. Previous research shows that maintenance of synaptic vesicle membrane protein levels is critical for synapse formation, neurotransmission and prevention of abnormal protein accumulation leading to neurodegeneration. Consistent with the NINDS mission "to reduce the burden of neurological disease", this proposal illuminates the role of endo-lysosomal transport in maintaining proper levels of synaptic vesicle proteins for neurotransmission while preventing the aberrant accumulation of synaptic vesicle proteins underlying neurodegeneration. [unreadable] [unreadable]